JP2009168671A - Biosensor system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor system for accurately quantifying a substrate of a dehydrogenase the coenzyme of which is a NAD<SP>+</SP>and a NADP<SP>+</SP>with no effect by a dissolved oxygen, inexpensively manufactured, and excellent in the portability. <P>SOLUTION: The biosensor of the invention is the biosensor system for detecting the substrate of the dehydrogenase the coenzyme of which is the NAD<SP>+</SP>and the NADP<SP>+</SP>, and includes: an optical fiber probe composed by connecting a light entering optical fiber and a light receiving optical fiber; an ultraviolet light-emitting diode for causing ultraviolet rays having a particular wavelength to enter into the light entering optical fiber; and a detector for detecting an fluorescence excited and generated by an incident light entering from the ultraviolet light-emitting diode through the light receiving optical fiber. A film is formed by immobilizing the dehydrogenase, and closely contacts the tip of the optical fiber probe. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a biosensor system, and more particularly to a biosensor system that detects a substrate of a dehydrogenase that uses NAD ⁺ or NADP ⁺ as a coenzyme.

Alcohol abuse is one of the most important issues facing our society, as alcohol is deeply related to human health, and overdose affects human relationships. For example, nonpatent literature 1-6).
In terms of health, for example, alcoholic liver diseases in developed countries are known to account for more than 50% of all chronic liver diseases. That is, since excessive intake of alcohol is detrimental to health, it is important to accurately measure the ethanol concentration from the clinical aspect, particularly in the analysis of blood, serum and urine.

Moreover, although the alcoholic beverage is manufactured through the fermentation process, in order to manage the quality of the alcoholic beverage in the fermentation process, the measurement of the ethanol concentration in the process is important. Thus, it is particularly important to accurately measure the alcohol concentration also from an economic viewpoint.
From the above viewpoint, a method for accurately and simply measuring ethanol is required.

  Conventionally, various alcohol determination methods have been reported. For example, a biosensor using an enzyme as a base is used as an alcohol sensor (for example, Non-Patent Documents 7 and 8). An alcohol oxidase immobilized ethanol sensor is one of the most common methods for quantifying alcohol. Such an alcohol sensor has an advantage that it can be measured easily and rapidly when compared with a chromatography method, a spectroscopic analysis method or a distillation method. Moreover, since the enzyme is used, it has the advantage that selectivity is high. However, since alcohol oxidase is used, there is a problem that the sensor characteristics depend on the amount of dissolved oxygen in the sample.

On the other hand, biosensors using nicotinamide adenine dinucleotide (NAD ⁺ ) -dependent alcohol dehydrogenase have been reported (for example, Non-Patent Documents 9 and 10). Such biosensors measure substrates using a ratio of NADH / NAD ⁺ , measured primarily by optical or electrochemical methods. Moreover, such a biosensor can be used not only for measuring the ethanol concentration but also for measuring the concentration of various substrates. In a conventional biosensor using nicotinamide adenine dinucleotide (NAD ⁺ ) -dependent alcohol dehydrogenase, a mercury lamp is used as a light source. However, mercury lamps are expensive and very heavy, making them unsuitable for portable use.

G.Heiss, N.J.Johnson, S.Reiland, C.E.Davis, H.A.Tyroler, Circulation 62 (1980) 116 M.G.Marmot, Int.J. Epidemiol. 13 (1984) 160 S. Linn, M. Carroll, C. Johnson, R.fulwood, W. Kalsbeek, R. Briefel, Am. J. Public Health 83 (1993) 811 R.D.Moore, T.A.Pearson, Medicine 65 (1986) 242 D.S.Sscovick, N.S.Weiss, N.Fox, Am J. Epidemiol. 123 (1986) 499 A.S.Santos, A.C.Pereira, N.Duran, L.T.Kubota, Electrochim. Acta. 52 (2006) 215 G. Wen, Y. Zhang, S. Shuang, C. Dong, M.M.F. Choi, Biosens. Bioelectron. 23 (2007) 121 L.V.Shkotova, A.P.Soldatkin, M.V.Gomchar, W. Schuhman, S.V.Dzyadevych, Materials Science and Engineering C 26 (2006) 411 C. Schelp, Th. Sceper, F. Buckmann, K.F.Reardon, Anal. Chem. Acta 255 (1991) 223 J. Manso, M.L.Mena, P.Y.Sedeno, J.M.Pingarron, Electrochem. Acta (2007), doi; 10.1016 / j.eleacta.2007.10.0003

Therefore, the object of the present invention is to accurately determine the substrate of dehydrogenase having NAD ⁺ or NADP ⁺ as a coenzyme without being affected by dissolved oxygen, and can be produced at low cost, and An object is to provide a biosensor system with excellent portability.

  As a result of repeated studies to achieve the above object, the present inventors have made an optical fiber probe in which a film formed by immobilizing a dehydrogenase is closely attached, and an ultraviolet light emitting diode (UV) that makes ultraviolet light incident on the optical fiber probe. -LED), by using a detector that detects the generated fluorescence excited by the incident light incident from the ultraviolet light-emitting diode, the knowledge that the above-mentioned purpose can be achieved is obtained, and the investigation is repeated based on the knowledge. As a result, the present invention has been completed.

The present invention has been made based on the above findings, a biosensor system for detecting a substrate of dehydrogenase and NAD ⁺ or NADP ⁺ coenzyme, the biosensor system, for light incident An optical fiber probe formed by connecting an optical fiber and an optical fiber for receiving light; an ultraviolet light emitting diode that injects ultraviolet light of a specific wavelength into the optical fiber for light incidence; and excitation by incident light incident from the ultraviolet light emitting diode A biosensor comprising a detector for detecting the generated fluorescence through the optical fiber for light reception, wherein a membrane formed by immobilizing the dehydrogenase is in close contact with the tip of the optical fiber probe A system is provided.

The specific wavelength is preferably 300 to 370 nm, and the wavelength of the fluorescence is preferably 450 to 510 nm.
Specific examples of the dehydrogenase include alcohol dehydrogenase, and the substrate includes ethanol.
The biosensor system has a bandpass filter between the optical fiber for light incidence and the optical fiber probe, and has a long wavelength transmission filter between the optical fiber for light reception and the optical fiber probe. It is preferable.
Further, the biosensor system has a band filter between the optical fiber for light incidence and the optical fiber probe, and has a band filter between the optical fiber for light reception and the optical fiber probe. May be.

According to the present invention, it is possible to accurately quantify the amount of dehydrogenase substrate using NAD ⁺ or NADP ⁺ as a coenzyme without being affected by dissolved oxygen. The biosensor system of the present invention can be manufactured at low cost and has excellent portability.

The present invention is described in detail below.
The biosensor system of the present invention is a biosensor system that detects a substrate of a dehydrogenase that uses NAD ⁺ or NADP ⁺ as a coenzyme,
An optical fiber probe formed by connecting an optical fiber for light incidence and an optical fiber for light reception;
An ultraviolet light emitting diode that makes ultraviolet light of a specific wavelength incident on the optical fiber for light incidence; and a detector that detects fluorescence generated by being excited by incident light incident from the light emitting diode through the optical fiber for light reception Including
A membrane formed by immobilizing the dehydrogenase is in close contact with the tip of the optical fiber probe.

The biosensor system of the present invention is a system for detecting a dehydrogenase substrate using NAD ⁺ (oxidized nicotinamide adenine dinucleotide) or NADP ⁺ (oxidized nicotinamide adenine dinucleotide phosphate) as a coenzyme. is there. The dehydrogenase used in the biosensor system of the present invention is not particularly limited as long as NAD ⁺ or NADP ⁺ is used as a coenzyme. For example, alanine dehydrogenase, alcohol dehydrogenase, aldehyde dehydration are used. Elementary enzyme, isocitrate dehydrogenase, uridine-5′-diphospho-glucose dehydrogenase, galactose dehydrogenase, formate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glycerol dehydrogenase, glycerol- 3-phosphate dehydrogenase, glucose dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, sarcosine dehydrogenase, sorbitol dehydrogenase, carbonic acid dehydrogenase, lactate dehydration Elementary enzyme, 3-hydroxybutyrate dehydrogenase, pyruvate dehydrogenase, fructose Scan dehydrogenase, 6-phosphofructokinase gluconate dehydrogenase, formaldehyde dehydrogenase, available mannitol dehydrogenase, malate dehydrogenase, leucine dehydrogenase and the like. In addition, there are many enzyme reactions involving dehydrogenase with NAD ⁺ or NADP ⁺ as a coenzyme, and it is useful as a measurement method in various fields. Furthermore, by using dehydrogenase compared to oxidase, There is also an advantage that the influence of dissolved oxygen in the sample can be avoided.
That is, the substrate that can be detected using the biosystem of the present invention is a compound that becomes a substrate for the dehydrogenase. For example, when alcohol dehydrogenase is used as a dehydrogenase, ethanol can be detected and quantified.

Depending on the substrate concentration, NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate) is generated by an enzyme reaction between a dehydrogenase, a substrate, and NAD ⁺ or NADP ⁺ . In the biosensor system of the present invention, the concentration of the substrate is measured by measuring the concentration of NADH or NADPH. In the present invention, the concentration of NADH or NADPH is measured by detecting fluorescence excited by ultraviolet light incident from an ultraviolet light emitting diode.

  Next, the biosensor system of the present invention will be described with reference to the drawings. FIG. 1 is a drawing schematically showing a biosensor system 10 of the present invention. The biosensor system of the present invention has an optical fiber probe 12 formed by connecting a light incident optical fiber 11 and a light receiving optical fiber 11 '. As such a probe 12, you may use what is marketed, for example, Ocean Optics Inc. A combination of 2 in 1 Optical fiber assembly (BIF600-UV / VIS) and F100-9209 (Ocean Optics Inc.), which is commercially available from (USA), can be used.

  To the optical fiber 11 for light incidence, an ultraviolet light-emitting diode 14 that enters ultraviolet rays having a specific wavelength is connected. In the biosensor system shown in FIG. 1, a band filter (band pass filter) 16 is connected between the ultraviolet light emitting diode 14 and the optical fiber probe 12. In the biosensor system of the present invention, since the ultraviolet light-emitting diode 14 is used as the light source, the apparatus can be simplified, compared with the case where a mercury lamp is used as the light source, and can be manufactured at a low cost. Can also be used.

The case of dehydrogenase using NAD ⁺ as a coenzyme will be described. NADH absorbs UV light of 340 nm, but NAD ⁺ does not absorb UV light of 340 nm. Is used for exciting ultraviolet rays having a wavelength of 300 to 370 nm, preferably around 340 nm. Therefore, as shown in FIG. 1, it is preferable that a band-pass filter 16 is connected between the ultraviolet light-emitting diode 14 and the optical fiber probe 12. The band-pass filter means a filter that transmits only light having a specific wavelength out of light from the light source. In the present invention, for example, a filter that passes ultraviolet rays of 330 to 350 nm is used. As such a bandpass filter, a commercially available filter can be used without particular limitation.

In the biosensor system shown in FIG. 1, a detector 20 that detects fluorescence generated by excitation by incident light incident from an ultraviolet light emitting diode 14 is connected to the optical fiber 11 ′ for receiving light. Specifically, a spectrophotometer is used as the detector 20. In the case of NADH, the excited fluorescence is 450 to 510 nm, more specifically, around 491 nm. Therefore, as shown in FIG. 1, a long wavelength transmission filter that transmits only fluorescence having a wavelength larger than a specific wavelength. A (high pass filter) 18 is preferably disposed. In FIG. 1, a long wavelength transmission filter that transmits fluorescence having a wavelength of 400 nm or more is used. As such a long wavelength transmission filter, a commercially available filter can be used without particular limitation.
In the biosensor system of FIG. 1, the long wavelength transmission filter (high pass filter) 18 is used, but a band filter may be used instead of the long wavelength transmission filter (high pass filter). In this case, for example, a bandpass filter that transmits only fluorescence having a wavelength of 450 to 510 nm is used.

  In the biosensor system shown in FIG. 1, a computer system 22 for analyzing data received by the detector 20 is connected. By connecting such a computer system 22, data analysis is easy. .

Next, the optical fiber probe 12 used in the biosensor system shown in FIG. 1 will be described. The optical fiber probe 12 is in close contact with a membrane formed by immobilizing a dehydrogenase at its tip.
A membrane formed by immobilizing a dehydrogenase will be described. In the present specification, hereinafter, an enzyme-immobilized membrane means a membrane formed by immobilizing a dehydrogenase.
The enzyme-immobilized membrane is obtained by immobilizing a dehydrogenase on a carrier that is a membrane material. As the carrier to be used, those conventionally used for immobilizing enzymes can be used without particular limitation. Examples of such a material include polytetrafluoroethylene, polydimethylsiloxane, polypropylene, polyethylene, polymethyl methacrylate, and polystyrene. Although the thickness of such a support | carrier is not specifically limited, Preferably it is 100 nm-200 micrometers, More preferably, it is 10 micrometers-100 micrometers.

The biosensor system of the present invention quantifies NAD ⁺ or NAD ⁺ concentration by fluorescence generated by excitation with excitation light incident from an ultraviolet light-emitting diode, thereby dehydrogenase using NAD ⁺ or NAD ⁺ as a coenzyme. It detects the substrate. Therefore, the substrate in the sample and the dehydrogenase in the enzyme-immobilized membrane must react in the presence of the coenzyme. As will be described later, the biosensor system of the present invention reacts with NAD ⁺ or NAD ⁺ as a coenzyme in a sample solution in order to react the enzyme with a substrate in the presence of NAD ⁺ or NAD ⁺ as a coenzyme. The coenzyme and the substrate need to react with the enzyme in the enzyme-immobilized membrane. Therefore, the carrier constituting the enzyme-immobilized membrane is preferably porous. There are no particular restrictions on the size of the pores of the carrier, but usually the diameter is about 0.1 to 1 μm, preferably about 0.2 μm. The porosity of the carrier is preferably 60 to 90%.

The enzyme-immobilized membrane is formed by immobilizing a dehydrogenase on a carrier, and is produced by mixing an enzyme with, for example, a polymer, coating the carrier, and drying. As such a polymer, those conventionally used for producing an enzyme-immobilized membrane can be used without any particular limitation. For example, the photofunctionality of SbQ is added to polyethylene glycol, polyvinyl alcohol, polyethylene glycol or polyvinyl alcohol. Examples thereof include PVA-SbQ, SPP-H13, and a polymer containing a phosphorylcholine group in which groups are combined.
Among these, when a copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC), which is a polymer containing a phosphorylcholine group, and another monomer is used, it is not necessary to perform a desalting treatment after immobilizing the enzyme. Therefore, it is preferable.
In addition, as a method for immobilizing a dehydrogenase on a carrier, a method by cross-linking glutaraldehyde may be used, but the method is not particularly limited in the present invention.

  Examples of the monomer used to produce such a copolymer include 2-ethylhexyl (meth) acrylate, methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, and butyl (meth) acrylate. , Amyl (meth) acrylate, hexyl (meth) acrylate, heptyl (meth) acrylate, octyl (meth) acrylate, nonyl (meth) acrylate, decyl (meth) acrylate, dodecyl (meth) acrylate, hexadecyl (meth) acrylate, octadecyl (Meth) acrylate, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, ethylene glycol (meth) acrylate, ethylene glycol methyl ether (meth) acrylate Poly (ethylene glycol) (meth) acrylate, poly (ethylene glycol) methyl ether (meth) acrylate, N-vinylpyrrolidone, vinyl acetate, γ-methacryloxypropyltrimethoxysilane, γ-methacryloxypropyltriethoxysilane, vinyltris ( 2-methoxyethoxy) silane, methyltrivinylmethyldiethoxysilane, and the like. Such a copolymer can be obtained by radical copolymerization of MPC and the above monomer.

  Examples of the copolymer include a copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2-ethylhexyl methacrylate (EHMA), and the copolymer is represented by the following general formula (1). A polymer having repeating units.

  In General formula (1), a is 0.1-0.9, Preferably it is 0.1-0.4. Moreover, b is 0.1-0.9, Preferably it is 0.6-0.9.

  In order to obtain a polymer having a repeating unit represented by the general formula (1), the reaction is carried out by a known method. As the solvent to be used, any solvent that can dissolve the monomer can be used without limitation. For example, water, methanol, ethanol, propanol, t-butyl alcohol, benzene, toluene, dimethylformamide, tetrahydrofuran, chloroform, And mixtures thereof.

  Further, as the polymerization initiator, a commonly used radical initiator can be used without particular limitation, for example, an aliphatic azo compound such as 2,2′-azobisisobutyronitrile, azobisvaleronitrile, Examples thereof include organic peroxides such as benzoyl peroxide, lauroyl peroxide, ammonium sulfate, and potassium sulfate.

The molecular weight of the polymer having a repeating unit represented by the general formula (1) is usually about 5,000 to 3,000,000.
In addition, as a polymer which has a repeating unit represented by General formula (1), you may use what is marketed, for example, Nippon Oil & Fats Co., Ltd. product, Lipidure (trademark) series etc. can be used. .

Next, a method for producing the enzyme-immobilized membrane will be described with reference to the drawings. FIG. 2 is a diagram schematically showing a method for producing an enzyme-immobilized film and an optical fiber probe used in the biosensor system of the present invention.
First, a mixed solution 32 of a polymer and a dehydrogenase is applied to the carrier 31. As the polymer, those described above are used, and those described above are also used as the dehydrogenase. Examples of the solvent for dissolving the polymer and dehydrogenase into a mixed solution include ethanol. The concentration of the dehydrogenase and polymer in the mixed solution is not particularly limited, but usually, the dehydrogenase is about 50 units per cm ² of the carrier, and the polymer is about 1 μL (concentration: 10% by weight) per cm ² of the carrier. It is preferable to use it. Next, the mixed solution 32 applied on the carrier is dried to immobilize the enzyme on the carrier 31 to obtain the enzyme-immobilized film 33. The drying is usually performed at a temperature of about 0 to 8 ° C. for 0.5 to 6 hours.

  Next, the adhesion of the enzyme-immobilized film to the tip of the optical fiber probe will be described. As is apparent with reference to FIG. 2, the enzyme-immobilized film 33 is brought into close contact with the tip of the optical fiber probe 14, and the enzyme-immobilized film 33 is fixed to the tip of the optical fiber probe 14 by, for example, silicon tube ring 35. To do.

Next, a method for detecting a compound that is a substrate for a dehydrogenase using the biosensor system of the present invention will be described with reference to FIG.
In order to detect a compound that is a substrate for a dehydrogenase using the biosensor system 10 of the present invention, the optical fiber probe 12 is immersed in a sample solution. This sample solution contains NAD ⁺ or NADP ^{+ which} is a coenzyme for dehydrogenase.

The above-described enzyme-immobilized membrane (a membrane formed by immobilizing a dehydrogenase) is in close contact with the tip of the optical fiber probe 12, and the substrate in the sample solution is combined with NAD ⁺ or NADP ⁺ that is a coenzyme. Then, it enters the enzyme-immobilized membrane, the dehydrogenase immobilized on the enzyme-immobilized membrane reacts with the substrate, and the coenzyme NAD ⁺ or NADP ⁺ changes to NADH or NADPH, respectively. The NADH or NADPH generated in this way is detected by fluorescence. The case of dehydrogenase using NAD ⁺ as a coenzyme will be described. Ultraviolet light with a central wavelength of 335 nm irradiated from the ultraviolet light-emitting diode 14 is passed through the band-pass filter 16 and only ultraviolet light with a wavelength of 330 to 350 nm is passed. The excited fluorescence is allowed to pass through the long wavelength transmission filter 18 and only the fluorescence having a wavelength of 400 nm or more is allowed to pass, and then the fluorescence intensity at a wavelength of 491 nm is measured by the detector (spectral altimeter) 20. Data measured by the detector 20 is analyzed by the computer 22.

The biosensor system of the present invention, NAD ⁺ or NADP ⁺ to detect the substrate and comprising a compound of dehydrogenases and coenzymes (quantitative) of the fluorescence intensity initially with NAD ⁺ or NADP ⁺ the standard solution was measured, the measurement of the sample was carried out after preparing a calibration curve, the fluorescence was measured for samples containing known amounts of NAD ⁺ or NADP ^+, from the amount of consumed NAD ⁺ or NADP ^+, contained in a sample Determine the amount of the compound that is the substrate for the dehydrogenase that is present.

  The biosensor system of the present invention uses an ultraviolet light emitting diode as a light source, and can be manufactured at a lower cost than a conventionally used mercury lamp. Further, since the ultraviolet light-emitting diode has a weight smaller than that of the mercury lamp, it can be used for portable use. The biosensor system of the present invention can be used in, for example, a microchemical substance analysis system (μTAS).

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.
Example 1
The biosensor system shown in FIG. 1 was produced using the following apparatus and the like.
Ultraviolet light emitting diode: UVTOP BL335 (manufactured by Sensor Electronic Technology, Inc.)
Optical fiber (optical fiber assembly): BF600-UV / VIS (manufactured by Ocean Optics Inc.)
Optical fiber probe: F100-9090 (manufactured by Ocean Optics Inc.)
Band filter: (Band pass wavelength: 330 to 350 nm, manufactured by Asahi Spectroscopy)
Long wavelength transmission filter: (cutoff wavelength: 400 nm, manufactured by Asahi Spectroscopy Co., Ltd.)
Optical fiber spectrometer: USB2000 and USB4000 (Ocean Optics Inc.)
USB2000 is a detector shown in FIG. 1, and USB4000 is connected to an ultraviolet light emitting diode.

Polytetrafluoroethylene (porosity 80%, hole size 0.2 μm, JGWP14225, Millipore) cut to 1 × 1 cm was firmly attached to the tip of the optical fiber probe using a silicone-O-ring. The optical fiber probe is immersed in a phosphate buffer so that it becomes 1, 2, 5, 10, 40, 100, 300, 500, 1000, 2000, 5000 micromol / L every 2 minutes. , NADH solution was dropped and the fluorescence intensity was monitored.
The graph of the measurement result of fluorescence intensity is shown in FIG. In FIG. 3, the horizontal axis represents time (minutes) and the vertical axis represents fluorescence intensity. As is apparent from FIG. 3, it can be seen that the fluorescence intensity increases with increasing NADH concentration. From this result, it became clear that NADH can be detected and quantified by using the biosensor system provided with the optical fiber probe.

Next, the relationship between the fluorescence intensity obtained as described above and the NADH concentration was examined, and a calibration curve was prepared. FIG. 4 is a calibration curve showing the relationship between NADH concentration and fluorescence intensity. The horizontal axis represents the logarithm of NADH concentration (micromol / L), and the vertical axis represents fluorescence intensity, which is apparent from FIG. As described above, the NADH concentration was measured using the biosensor system equipped with the optical fiber probe, and the calibration curve was created. As a result, it was found that the NADH concentration showed a straight line in the range of 1 to 300 micromol / L. It was. That is, when the NADH concentration is in the above range, it was found that NADH can be quantified by the biosensor described above.
The calibration curve shown in FIG. 4 was expressed by the following equation, and the correlation coefficient was 0.993.
Fluorescence intensity (count) = 1.97 × [NADH (micromol / L)] ^0.89

Example 2
An enzyme-immobilized membrane was prepared by the following operation.
A mixed solution of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2-ethylhexyl methacrylate copolymer (EHMA) and alcohol dehydrogenase (solvent: ethanol) is 1 microliter / cm ² and 50 units / cm ² , respectively. Thus, it apply | coated on the polytetrafluoroethylene film | membrane used in Example 1, and left still for 3 hours in a refrigerator (about 4 degreeC), and the said mixed solution was hardened. Next, unnecessary alcohol dehydrogenase was washed with a phosphate buffer to obtain a membrane on which the dehydrogenase was immobilized.
The obtained film was firmly adhered to the tip of the optical fiber probe in the biosensor system shown in FIG. 1 using a silicone-O-ring to obtain the biosensor system of the present invention.

Next, the optical fiber probe is immersed in a phosphate buffer containing NAD ⁺ (concentration of 20 mmol / liter), and every 2, 2 minutes, 0.1, 1, 5, 10, 50, 100, 500, 1000, respectively. Ethanol was added dropwise to monitor the fluorescence intensity.
The graph of the measurement result of fluorescence intensity is shown in FIG. In FIG. 5, the horizontal axis represents time (minutes), and the vertical axis represents fluorescence intensity. As can be seen from FIG. 3, as the ethanol concentration increases, the fluorescence intensity increases accordingly. From this result, it was revealed that ethanol can be detected and quantified by using the biosensor system of the present invention provided with the above optical fiber probe.

Next, the relationship between the fluorescence intensity obtained as described above and the ethanol concentration was examined to prepare a calibration curve. FIG. 6 is a calibration curve showing the relationship between the ethanol concentration and the fluorescence intensity. The horizontal axis represents the logarithm of the ethanol concentration (mmol / L), and the vertical axis represents the fluorescence intensity. As apparent from FIG. In addition, the concentration of ethanol was measured using the biosensor system of the present invention and a calibration curve was created. As a result, it was found that the ethanol concentration showed a straight line in the range of 1 to 100 mmol / L. That is, it was found that when the ethanol concentration is in the above range, ethanol can be quantified by the biosensor of the present invention.
The calibration curve shown in FIG. 6 was expressed by the following equation, and the correlation coefficient was 0.994.
Fluorescence intensity (count) = 1.54 × [ethanol (mmol / L)] ^0.52

  Since the biosensor system produced in Example 2 uses an ultraviolet light emitting biode as a light source, it is lighter in weight than a sensor using a conventional mercury lamp, and is therefore suitable for portable use. Further, when ethanol was quantified using the biosensor system of Example 2, the total power consumption was about 150 mW, which was about 1% of the case where a conventional mercury lamp was used. Moreover, since the biosensor system of the present invention is lightweight, it can be used, for example, in a microchemical substance analysis system (μTAS).

It is drawing which shows typically the biosensor system of this invention. It is a figure which shows roughly the manufacturing method of the enzyme fixed film | membrane and optical fiber probe used in the biosensor system of this invention. It is a graph which shows the measurement result of fluorescence intensity. 2 is a calibration curve showing the relationship between NADH concentration and fluorescence intensity. It is a graph which shows the measurement result of fluorescence intensity. 2 is a calibration curve showing the relationship between ethanol concentration and fluorescence intensity.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Biosensor system 11 Optical fiber 11 'for light incidence Optical fiber 11' for light reception 12 Optical fiber probe 14 Ultraviolet light emitting diode
16 Band pass filter (band filter)
18 Long wavelength transmission filter (high pass filter)
20 detector 22 computer system 31 carrier 32 mixed solution of polymer and dehydrogenase 33 enzyme-immobilized film 34 optical fiber probe 35 silicon tube ring

Claims (5)

A biosensor system for detecting a substrate of a dehydrogenase having NAD ⁺ or NADP ⁺ as a coenzyme,
The biosensor system includes:
An optical fiber probe formed by connecting an optical fiber for light incidence and an optical fiber for light reception;
An ultraviolet light-emitting diode that injects ultraviolet light of a specific wavelength into the optical fiber for light incidence; and a detector that detects fluorescence generated by excitation by incident light incident from the ultraviolet light-emitting diode through the optical fiber for light reception Including
A biosensor system, wherein a membrane formed by immobilizing the dehydrogenase is in close contact with the tip of the optical fiber probe. The biosensor system according to claim 1, wherein the specific wavelength is 300 to 370 nm, and the wavelength of the fluorescence is 450 to 510 nm. The biosensor system according to claim 1 or 2, wherein the dehydrogenase is alcohol dehydrogenase and the substrate is ethanol. 4. A band-pass filter is provided between the optical fiber for light incidence and the optical fiber probe, and a long wavelength transmission filter is provided between the optical fiber for light reception and the optical fiber probe. The biosensor system according to any one of the above. The bandpass filter is provided between the optical fiber for light incidence and the optical fiber probe, and the bandpass filter is provided between the optical fiber for light reception and the optical fiber probe. The biosensor system according to claim 1.

Images